The main goal of this project is to study the role of novel T cell costimulatory pathways in regulating alloimmune responses in vivo. We have a number of unique tools that will enable us to dissect the functions, mechanisms, and interactions of newly discovered T cell costimulatory pathways in regulating alloimmune responses in vivo. The proposal has two major specific aims. In aim 1, we plan to investigate the functions of novel T-cell costimulatory pathways in regulating alloimmune responses in vivo using models of solid organ and tissue (skin) transplants. Initially, we will focus on studying the functions of the PD1-PDLI/PDL2 pathway in regulating alloimmune responses in vivo. In future years we plan to study the functions of newer pathways as they are initially identified and appropriate reagents and animals are generated by the cores of the overall program project. Our hypothesis is that the PD1-PDLI/PDL2 pathway negatively regulates alloimmune responses in vivo. We will use gene targeting approaches and blocking monoclonal antibodies as tools to investigate the functions of this new pathway in alloimmune responses in vivo. We will define the role of this pathway in regulating CD4+ and CD8+ alloreactive T cells, Thl and Th2 alloimmune responses, and acute and chronic rejection using established skin and vascularized cardiac transplantation models in mice. We will also define the interactions between the PD1 pathway and other CD28 homologues (CD28, CTLA4, ICOS). Our corollary hypothesis is that signaling through PD1 promotes tolerance in alloreactive T cells. We will test novel therapeutic combinations using a PDL1Fc to promote donor specific tolerance. We will use unique in vitro and in vivo assays including specific TCR transgenic animals with defined allospecificity to class I (CD8) or class II MHC (CD4) alloantigens to better understand the mechanisms of targeting the PD1 costimulatory pathway in vivo. In aim 2 we will focus on developing non-toxic strategies to achieve donor bone marrow chimerism across fully allogeneic barriers without chemoradiotherapy conditioning. Our hypothesis is that targeting T cell costimulatory pathways promotes development of mixed allogeneic chimerism without chemoradiotherapy. We will target well defined and new T costimulatory pathways that are known to regulate initial CD4+ and CD8+ T cell expansion and/or subsequent survival. Successful strategies that induce donor T cell chimerism will be tested for specificity of tolerance using donor and third party skin graft rejection as a stringent transplant model. Furthermore, using novel models permitting tracking of host anti-donor alloreactive CD4+ and CD8+ TCR transgenic T cells, we will examine the biology of graft resistance and determine how targeting new T cell costimulatory pathways affect alloreactivity in the context of mixed allogeneic chimerism. Overall, the studies in this project will dissect the functions and mechanisms of novel T cell costimulatory pathways in allograft rejection and tolerance. The results should yield useful information for future development of novel therapeutic strategies to induce transplantation tolerance to translate to primates and humans.